Conditional superagonist CTL ligands for the promotion of tumor-specific CTL responses

ABSTRACT

What is described is a novel genetic screen, involving recombinant technology and class I antigen cross-presentation, to search for supraoptimal superagonists of the 27L MART-1 mutant selecting for single amino acid substitution mutants of 27L that activate human antigen-specific CTL clones recognizing the wild-type MART-1 26-35  epitope. Three novel mutant epitopes are identified with superagonist properties that are functionally superior to 27L. The ability of a given analog to act as superagonist varies among patients. Also described is the use of methods to establish panels of potential superagonist APLs to individualize tumor peptide vaccines among patients. The methodology is replicated to identify APL to NY-ESO-1 157-165  and NY-ESO-1 157-170  tumor epitopes. A general method is described that is useful to produce a tumor vaccine to any tumor epitope.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. 371 ofInternational Application Number PCT/US2011/035272, filed May 4, 2011,which claims priority to U.S. provisional application No. 61/331,260,filed May 4, 2010, which is hereby incorporated by reference in itsentirety.

This invention was made with government support under CA122904 awardedby the National Institutes of Health and National Cancer Institute. Thegovernment has certain rights to the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 11, 2014, isnamed 105003.000128_SL.txt and is 69,434 bytes in size.

TECHNICAL FIELD

What is described is a method of identifying antigens for a cancervaccine, and specific polypeptides and polynucleotides useful inproducing vaccines against cells having NY-ESO-1₁₅₇₋₁₆₅,NYE-ESO-1₁₅₇₋₁₇₀, or MART-1₂₆₋₃₅ tumor epitopes.

BACKGROUND

Cytotoxic T lymphocytes can directly kill malignant cells, which expressand display specific antigenic peptides in the context of specific classI MHC molecules. These antigenic peptides, often referred to as CTLepitopes, are peptides of unique amino acid sequence, usually 9-11 aminoacids in length. The tumor-associated antigenic peptide that is beingtargeted can be used as a peptide-based vaccine to promote theanti-tumor CTL response. However, when the target peptide is derivedfrom non-mutated differentiation antigens as is often the case (e.g.melanosomal proteins), it can be insufficient to engender robust andsustained anti-tumor CTL responses. This is a result of immune tolerancemechanisms that generally suppress or eliminate high avidityauto-reactive T cells. As a result of these mechanisms, the vastmajority of tumor-specific CTL, specifically those that recognizenon-mutated tumor-associated antigens, are eliminated in the thymus andin the periphery. What remains is a low frequency of tumor-specific CTL,and/or CTL that bear low avidity T cell receptors for the cognate tumorantigen.

One way to activate and mobilize these rare and low aviditytumor-specific CTL is with the use of superagonist altered peptideligands (APLs). These are mutant peptide ligands that deviate from thenative peptide sequence by one or more amino acids, and which activatespecific CTL clones more effectively than the native epitope. Thesealterations either allow the peptide to bind better to the restrictingclass I MHC molecule or interact more favorably with the TCR of a giventumor-specific CTL subset. Superagonist APLs demonstrate favorableresponses in clinical studies.

One method to identify superagonist APLs involves comparing the aminoacid sequence of the tumor-associated CTL epitope to the so-calledconsensus binding motif for the restricting class I MHC allotype. Wherethe tumor-associated epitope deviates from the consensus sequence, theappropriate amino acids can be substituted, allowing the peptide to bindbetter to the class I MHC molecule. This approach is limited because notall poorly stimulatory CTL epitopes deviate from the consensus motif.Another approach involves substituting one or more specific amino acidsinto every position of the epitope; e.g., alanine scanning. Anotherapproach includes making every single amino acid substitution at one ortwo positions—positions either predicted to play a role in class I MHCsecondary binding or to be directly involved in engaging the TCR. All ofthese approaches are severely limited in scope, and potentially overlooka large number of superagonist APLs. Utilization of APLs remains limiteddue to a lack of comprehensive methods for which to identify them.

SUMMARY

One aspect of the invention is a method of identifying an alteredpeptide ligand (APL) for eliciting response of regulator or effector CD4or CD8 T cells against a tumor epitope, consisting of the steps of:

-   -   i. preparing saturation mutagenesis oligonucleotides encoding        APLs;    -   ii. cloning said oligonucleotides in an expression vector;    -   iii. separating APL expressed by a clone from other cellular        proteins;    -   iv. treat the T cells with the APL; and    -   v. identifying a superagonist APL that maximally stimulates the        T cells.

An embodiment of the method, is a further step of screening the APLs forthe ability to activate epitope-specific CTL clones. Another embodimentis the method in which the APLs are cross-presented to CTL clones onclass I MHC molecules by immature dendritic cells. Another embodiment isthe method wherein the oligonucleotides are cloned into bacteria.Another embodiment is wherein the bacteria are grown in 5 ml cultures,preferably less than 1 ml and preferably more than 0.2 ml. Another isthe method, wherein the APL stimulates T cells to produce interferon-γ.

Another aspect of the method is wherein the tumor epitope is selectedfrom the group consisting of SEQ ID NOS:1-351. An embodiment of themethod is wherein the tumor epitope is NYESO-1₁₅₇₋₁₆₅ (SEQ ID NO:366),NYESO-1₁₅₇₋₁₇₀ (SEQ ID NO:367) or MART-1₂₆₋₃₅ (SEQ ID NO:361). Anotherembodiment is the superagonist APL identified by use of these tumorepitopes. Another embodiment is the method, wherein a panel ofsuperagonist APLs are identified.

Another aspect of the invention is the superagonist APL identifiedaccording to the method, including SEQ ID NOS:362-365 and 368-375. Oneor more APL is useful as a component in an antitumor vaccine. Anembodiment is a panel of superagonist APLs, consisting of two or moreAPLs.

Another aspect of the invention is a method of using the superagonistAPL by combining it with cells of a patient. An embodiment is a methodby which the superagonist APL is administered to the patient. In thiscontext, the superagonist APL may be used in an anti-tumor vaccine, inadoptive immunotherapy to generate T cell clonotypes, and/or to alterthe phenotype of regulatory T cells to more effectively activateanti-tumor T cells. This embodiment of the invention may involve cellsof the patient being treated ex vivo.

Another aspect of the invention is the method of stimulating T cellswith an APL or APL panel, wherein the CD4 T cells are one or more Tcells selected from the group consisting of Th1, Th2, Th9, and Th17cells.

Another aspect of the invention is a superagonist APL selected from thegroup consisting of SEQ:362-365 and 368-376. An embodiment of theinvention is a panel of superagonist APLs selected from the groupconsisting of SEQ:362-365 and 368-376.

Another aspect of the invention is an APL minigene, comprising anucleotide sequence selected from the group consisting of SEQ IDNO:382-391 and 377-381. An embodiment of the invention is a use of theAPL minigene to express the minigene in a human cell, preferably a cellof a patient, most preferably, a cell from a patient with a cancer orcancer precursor, a graft versus host disease, or an autoimmunecondition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Native and superagonist CTL determinants can be distinguished inbead-based cross presentation assay. Oligonucleotides encodingMART-1₂₆₋₃₅, NY-ESO-1₁₅₇₋₁₆₅, or MART-1₂₆₋₃₅A27L were cloned into andexpressed by pQE40 expression vectors in 5 ml bacterial cultures. Themini-gene products were isolated and “fed” to immature dendritic cellsas described in the Examples. MART-1₂₆₋₃₅-specific CTL clones were usedto detect the presence of the cross-presented mini-gene products.Induced IFN-γ expression was determined by standard sandwich ELISA. A27Lsynthetic peptide (SEQ ID NO:362) at 1 μM was used a positive control.

FIG. 2. Previously described superagonists identified in MART-1₂₆₋₃₅Position 2 saturation mutagenesis APL screen. 88 P2 saturationmutagenesis clones were screened. MART-1₂₆₋₃₅ control construct is thefirst bar on left, and the NY-ESO-1₁₅₇₋₁₆₅ negative control construct isthe second bar from left. APL clones eliciting comparable IFN-γexpression as the native construct were sequenced. The amino acid atposition 27 is shown above for the most active polypeptide sequences.

FIG. 3. Eight positional libraries of A27L were screened using thesaturation mutagenesis technique. 88 mutant clones were screened foreach of eight positional libraries of A27L-P1, P3, P4, P5, P6, P7, P8and P9. (Fin. 3A) 2 clones are screened simultaneously for each library.Activation was assessed by IFN-γ expression. Positive control (A27L; SEQID NO:362) is the far left bar while the negative control(NYESO-1₁₅₇₋₁₆₅) is the second from left. APL clonal wells indicatedwith an arrow were de-convoluted and each mutant APL re-screenedseparately. FIG. 3B shows the IFN-γ activity elicited by individualclones, relative to the activity elicited by A27L. The clones that wereinitially assayed together are indicated by shading. A bold numberindicates the APL clone which is most responsible for the activation ofthe screening CTL clone. DNA sequence analysis was used to determine theamino acid encoded.

FIG. 4. APLs identified in saturation mutagenesis screen activate uniqueMART-1₂₆₋₃₅-specific CTL clones differently. Two unique high avidityMART-1₂₆₋₃₅-specific CTL clones, M26-H1 (A) and M26-H2 (B), and twounique low avidity MART-1₂₆₋₃₅-specific CTL clones, M26-L1(C) and M26-L2(D), were assayed against the agonist peptides A27L (square), E26G (SEQID NO:363; circle), E26S (SEQ ID NO:364; triangle), L33M (SEQ ID NO:365;diamond) and NY-ESO-1₁₅₇₋₁₆₅ (x's). Peptides were titrated on T2 targetcell. IFN-γ expression was measured by standard ELISA.

FIG. 5. APLs generate different CTL responses from the PBMC of differentmelanoma patients. Identified APLs were used to stimulate peripheralblood mononuclear cells (PBMC) of different melanoma patients in vitro.Following a one week primary and one week secondary peptide stimulation,cultures were stained with FITC-labeled anti-CD8 antibody andAPC-labeled HLA-A2/MART-1₂₆₋₃₅ tetramer and analyzed by flow cytometry.Data is representative of at least three different experiments.

FIG. 6. Native and superagonist CTL determinants can be distinguished inbead-based cross-presentation assay. Oligonucleotides encodingNY-ESO-1₁₅₇₋₁₇₀ were cloned into and expressed by pQE40 expressionvectors. The mini-gene products were isolated and “fed” to immaturedendritic cells as described in Examples 2-8. NY-ESO-1₁₅₇₋₁₇₀₋specificCTL clones were used to detect the presence of the cross-presentedmini-gene products. Induced IFN-γ expression was determined by standardsandwich ELISA. Synthetic wild-type peptide was used a positive control.

FIG. 7. NYESO-1₁₅₇₋₁₆₅ C165V generates Specific CTL better than the wildtype peptide. Following a 1-week primary and 1-week secondary peptidestimulation (Week 2), and an additional week (Week 3), cultures werestained with FITC-labeled anti-CD8 antibody and APC-labeledHLA-A2/NY-ESO and analyzed by flow cytometry. Two peptides were tested,NY-ESO-I₁₅₇₋₁₆₅ (SEQ ID NO:366) wild-type, and NY-ESO-1₁₅₇₋₁₇₀-(V) (SEQID NO:376).

FIG. 8. Native and superagonist CTL determinants can be distinguished inbead-based cross presentation assay. Oligonucleotides encodingNY-ESO-1₁₅₇₋₁₇₀ were cloned into and expressed by pQE40 expressionvectors. The mini-gene products were isolated and “fed” to immaturedendritic cells as described in the Examples. NY-ESO-1₁₅₇₋₁₇₀-specificCTL clones were used to detect the presence of the cross-presentedmini-gene products (NY-ESO-1₁₅₇₋₁₇₀). PBMC (FIG. 8B) were compared toCD-4⁺ cells (FIG. 8A). Induced IFN-γ expression was determined bystandard sandwich ELISA. Synthetic wild-type peptide was used a positivecontrol. The designations for the clones are as follows: NYII WT-1 isSEQ ID NO:362; NYII-5I-1 and -2 are W161I (SEQ ID NO:368); NYII-6Q-1 and-2 are I162Q (SEQ ID NO:372); NYII-6V-1 and -2 are I162V (SEQ IDNO:373); NYII-8S-1 and -2 are Q164S (SEQ ID NO: 374); NYII14W-1 and -2are F170W (SEQ ID NO:375).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

What is described herein is a method to screen for potentialsuperagonist APLs of a clinically relevant tumor-associated antigen,including NY-ESO-1 and MART-1. Rather than screening a limited subset ofpossible agonists, this technique allows screening of every single aminoacid mutant of tumor epitope in a rapid and cost-effective manner. Thisapproach to identifying APLs is effective, given the difference evensubtle amino acid substitutions have on specific T cell response. Sincesuperagonist APL structure cannot be predicted, the method describedgenerates candidate APLS by a comprehensive screening technique.

Another aspect of unpredictability is that a given agonist APL may bemore or less effective for different patients. While a given agonist APLmight have a high stimulatory capacity for one patient it could berelatively ineffective for another patient. Apparently, different clonesare being mobilized with different agonist peptides. This heightens theneed for panels of superagonist APLs for use in a therapeutic setting.

Tumor-Specific Epitopes

Unique antigens result from point mutations in genes that are expressedubiquitously. The mutation usually affects the coding region of the geneand is unique to the tumor of an individual patient or restricted tovery few patients. Antigens that are strictly tumor-specific may play animportant role in the natural anti-tumor immune response of individualpatients. These are listed in Table 1.

These epitopes are characteristic of lung carcinoma, melanoma, chronicmyeloid leukemia, colorectal carcinoma, gastric carcinoma, endometrialcarcinoma, head and neck squamous cell carcinoma, lung squamous cellcarcinoma, renal cell carcinoma, bladder tumor, non-small cell lungcarcinoma, head and neck squamous cell carcinoma, pancreaticadenocarcinoma, sarcoma, promyelocytic leukemia, myeloid leukemia, acutelymphoblastic leukemia, acute myelogenous leukemia, breast cancer, andprostate carcinoma.

Each epitope is associated with a particular HLA haplotype, either aclass I or class II MHC antigen, as shown in Tables 1-4.

TABLE 1 Unique antigens SEQ ID Gene/protein HLA haplotype Peptide^(c)Position NO alpha-actinin-4 A2 FIASNGVKLV  118-127 1 ARTC1 DR1YSVYFNLPADTIYTN 2 BCR-ABL fusion protein A2 SSKALQRPV  926-934 3 (b3a2)B8 GFKQSSKAL  922-930 392 DR4 ATGFKQSSKALQRPVAS  920-936 393 DR9ATGFKQSSKALQRPVAS  920-936 394 B-RAF DR4 EDLTVKIGDFGLATEKSRWSGSHQFEQLS 586-614 4 CASP-5 A2 FLIIWQNTM   67-75 5 CASP-8 B35 FPSDSWCYF  476-484 6beta-catenin A24 SYLDSGIHF   29-37 7 Cdc27 DR4 FSWAMDLDPKGA  760-771 8CDK4 A2 ACDPHSGHFV   23-32 9 CDKN2A A11 AVCPWTWLR  125-133 10(p14ARF-ORF3)  111-119 (p16INK4a- ORF3) COA-1 DR4 TLYQDDTLTLQAAG 371-384 11 DR13 TLYQDDTLTLQAAG  371-384 413 dek-can fusion protein DR53TMKQICKKEIRRLHQY  342-357 12 EFTUD2 A3 KILDAVVAQK  668-677 13Elongation factor 2 A68 ETVSEQSNV  581-589 14 ETV6-AML1 A2 RIAECILGM 334-342 15 fusion protein DP5 IGRIAECILGMNPSR  332-346 16 DP17IGRIAECILGMNPSR  332-346 414 FLT3-ITD A1 YVDFREYEYY  591-600 17 FN1 DR2MIFEKHGFRRTTPP 2050-2063 18 GPNMB A3 TLDWLLQTPK  179-188 19LDLR-fucosyltransferase DR1 WRRAPAPGA  315-323 20 AS fusion protein DR1PVTWRRAPA  312-320 21 hsp70-2 A2 SLFEGIDIYT  286-295 22 KIAAO205 B44AEPINIQTW  262-270 23 MART2 A1 FLEGNEVGKTY  446-455 24 ME1 A2 FLDEFMEGV 224-232 25 MUM-1^(f) B44 EEKLIVVLF   30-38 26 MUM-2 B44 SELFRSGLDSY 123-133 27 Cw6 FRSGLDSYV  126-134 28 MUM-3 A68 EAFIQPITR  322-330 29neo-PAP DR7 RVIKNSIRLTL  724-734 30 Myosin class I A3 KINKNPKYK  911-91931 NFYC B52 QQITKTEV  275-282 32 OGT A2 SLYKFSPFPL   28-37 33 OS-9 B44KELEGILLL  438-446 34 p53 A2 VVPCEPPEV  217-225 35 pml-RAR alpha fusionDR11 NSNHVASGAGEAAIETQSSSSEEIV 36 protein PRDX5 A2 LLLDDLLVSI  163-17237 PTPRK DR10 PYYFAAELPPRNLPEP  667-682 38 K-ras B35 VVVGAVGVG    7-1539 N-ras A1 ILDTAGREEY   55-64 40 RBAF600 B7 RPHVPESAF  329-337 41 SIRT2A3 KIFSEVTLK  192-200 42 SNRPD1 B38 SHETVIIEL   11-19 43SYT-SSX1 or -SSX2 B7 QRPYGYDQIM  402-410 (SYT) 44 fusion protein 111-112(SSX2) TGF-betaRII A2 RLSSCVPVA  131-139 45 Triosephosphate DR1GELIGILNAAKVPAD   23-37 46 isomerase

Shared antigens are present on many independent tumors. One groupcorresponds to peptides encoded by “cancer-germline” genes that areexpressed in many tumors but not in normal tissues. Some are listed inTable 2.

TABLE 2 Shared tumor-specific antigens Gene HLA Peptide PositionSEQ ID NO: 4BAGE-1 Cw16 AARAVFLAL   2-10 47 GAGE-1,2,8 Cw6 YRPRPRRY  9-16 48 GAGE-3,4,5,6,7 A29 YYWPRPRRY  10-18 49 GnTV^(f) A2VLPDVFIRC(V) intron 50 HERV-K-MEL A2 MLAVISCAV   1-9 51 KK-LC-1 B15RQKRILVNL  76-84 52 KM-HN-1 A24 NYNNFYRFL 196-204 53 A24 EYSKECLKEF499-508 54 A24 EYLSLSDKI 770-778 55 LAGE-1 A2 MLMAQEALAFL ORF2 56 (1-11)A2 SLLMWITQC 157-165 57 A31 LAAQERRVPR ORF2 58 (18-27) A68 ELVRRILSR103-111 59 B7 APRGVRMAV ORF2 60 (46-54) DP4 SLLMWITQCFLPVF 157-170 61DR3 QGAMLAAQERRVPRAAEVPR ORF2 62 (14-33) DR4 AADHRQLQLSISSCLQQL 139-15663 DR11 CLSRRPWKRSWSAGSCPGMPHL ORF2 64 (81-102) DR12CLSRRPWKRSWSAGSCPGMPHL ORF2 65 (81-102) DR13 ILSRDAAPLPRPG 108-120 66DR15 AGATGGRGPRGAGA  37-50 67 MAGE-A1 A1 EADPTGHSY 161-169 68 A2KVLEYVIKV 278-286 69 A3 SLFRAVITK  96-104 70 A68 EVYDGREHSA 222-231 71B7 RVRFFFPSL 289-298 72 B35 EADPTGHSY 161-169 73 B37 REPVTKAEML 120-12974 B53 DPARYEFLW 258-266 75 B57 ITKKVADLVGF 102-112 76 Cw2 SAFPTTINF 62-70 77 Cw3 SAYGEPRKL 230-238 78 Cw16 SAYGEPRKL 230-238 79 DP4TSCILESLFRAVITK  90-104 80 DP4 PRALAETSYVKVLEY 268-282 81 DR13FLLLKYRAREPVTKAE 112-127 82 DR15 EYVIKVSARVRF 281-292 83 MAGE-A2 A2YLQLVFGIEV 157-166 84 A24 EYLQLVFGI 156-164 85 B37 REPVTKAEML 127-136 86Cw7 EGDCAPEEK 212-220 87 DR13 LLKYRAREPVTKAE 121-134 88 MAGE-A3 A1EVDPIGHLY 168-176 89 A2 FLWGPRALV 271-279 90 A2 KVAELVHFL 112-120 91 A24TFPDLESEF  97-105 92 A24 VAELVHFLL 113-121 93 B18 MEVDPIGHLY 167-176 94B35 EVDPIGHLY 168-176 95 B37 REPVTKAEML 127-136 96 B40 AELVHFLLL 114-12297 B44 MEVDPIGHLY 167-176 98 B52 WQYFFPVIF 143-151 99 Cw7 EGDCAPEEK212-220 100 DP4 KKLLTQHFVQENYLEY 243-258 101 DQ6 KKLLTQHFVQENYLEY243-258 102 DR1 ACYEFLWGPRALVETS 267-282 103 DR4 VIFSKASSSLQL 149-160104 DR7 VIFSKASSSLQL 149-160 105 DR11 GDNQIMPKAGLLIIV 191-205 106 DR11TSYVKVLHHMVKISG 281-295 107 DR13 RKVAELVHFLLLKYRA 111-126 108 DR13FLLLKYRAREPVTKAE 119-134 109 MAGE-A4 A1 EVDPASNTY 169-177 110 A2GVYDGREHTV 230-239 111 A24 NYKRCFPVI 143-151 112 B37 SESLKMIF 156-163113 MAGE-A6 A34 MVKISGGPR 290-298 114 B35 EVDPIGHVY 168-176 115 B37REPVTKAEML 127-136 116 Cw7 EGDCAPEEK 212-220 117 Cw16 ISGGPRISY 293-301118 DR13 LLKYRAREPVTKAE 121-134 119 MAGE-A9 A2 ALSVMGVYV 223-231 120MAGE-A10 A2 GLYDGMEHL 254-262 121 B53 DPARYEFLW 290-298 122 MAGE-A12A2^(g) FLWGPRALV 271-279 123 Cw7 VRIGHLYIL 170-178 124 Cw7 EGDCAPEEK212-220 125 DP4 REPFTKAEMLGSVIR 127-141 126 DR13 AELVHFLLLKYRAR 114-127127 MAGE-C2 A2 LLFGLALIEV 191-200 128 A2 ALKDVEERV 336-344 129 B44SESIKKKVL 307-315 130 mucin ^(k) PDTRPAPGSTAPPAHGVTSA 131 NA88-A B13QGQHFLQKV 132 NY-ESO-1/LAGE-2 A2 SLLMWITQC 157-165 133 A2 MLMAQEALAFLORF2 134 (1-11) A31 ASGPGGGAPR  53-62 135 A31 LAAQERRVPR ORF2 136(18-27) A68 TVSGNILTIR 127-136 137 B7 APRGPHGGAASGL  60-72 138 B35MPFATPMEA  94-102 139 B49 KEFTVSGNILTI 124-135 140 B51 MPFATPMEA  94-102141 Cw3 LAMPFATPM  92-100 142 Cw6 ARGPESRLL  80-88 143 DP4SLLMWITQCFLPVF 157-170 144 DP4 LLEFYLAMPFATPMEAELARRSLAQ  87-111 145 DR1LLEFYLAMPFATPMEAELARRSLAQ  87-111 146 DR1 EFYLAMPFATPM  89-100 147 DR2RLLEFYLAMPFA  86-97 148 DR3 QGAMLAAQERRVPRAAEVPR ORF2 149 (14-33) DR4PGVLLKEFTVSGNILTIRLT 119-138 150 DR4 VLLKEFTVSG 121-130 151 DR4AADHRQLQLSISSCLQQL 139-156 152 DR4 LLEFYLAMPFATPMEAELARRSLAQ  87-111 153DR7 PGVLLKEFTVSGNILTIRLTAADHR 119-143 154 DR7 LLEFYLAMPFATPMEAELARRSLAQ 87-111 155 DR15 AGATGGRGPRGAGA  37-50 156 SAGE A24 LYATVIHDI 715-723157 Sp17 A1 ILDSSEEDK 103-111 158 SSX-2 A2 KASEKIFYV  41-49 159 DP1EKIQKAFDDIAKYFSK  19-34 160 DR3 WEKMKASEKIFYVYMKRK  37-54 161 DR4KIFYVYMKRKYEAMT  45-59 162 DR11 KIFYVYMKRKYEAM  45-58 163 SSX-4 DP10INKTSGPKRGKHAWTHRLRE 151-170 164 DR3 YFSKKEWEKMKSSEKIVYVY  31-50 165 DR8MKLNYEVMTKLGFKVTLPPF  51-70 166 DR8 KHAWTHRLRERKQLVVYEEI 161-180 167DR11 LGFKVTLPPFMRSKRAADFH  61-80 168 DR15 KSSEKIVYVYMKLNYEVMTK  41-60169 DR52 KHAWTHRLRERKQLVVYEEI 161-180 170 TAG-1 A2 SLGWLFLLL  78-86 171B8 LSRLSNRLL  42-50 172 TAG-2 B8 LSRLSNRLL  42-50 173 TRAG-3 DR1CEFHACWPAFTVLGE  34-48 174 DR4 CEFHACWPAFTVLGE  34-48 175 DR7CEFHACWPAFTVLGE  34-48 176 TRP2-INT2^(g) A68 EVISCKLIKR intron 2 177XAGE-1b DR9 CATWKVICKSCISQTPG  33-49 178

A second group of shared tumor antigens, named differentiation antigens,are also expressed in the normal tissue of origin of the malignancy.Antigens of this group are not tumor-specific, and their use as targetsfor cancer immunotherapy may result in autoimmunity towards thecorresponding normal tissue. Autoimmune toxicity should not be an issue,however, in situations where the tissue expressing the antigen isdispensable or even resected by the surgeon in the course of cancertherapy, as would be the case for prostate specific antigen (PSA). Theseantigens are listed in Table 3.

TABLE 3 Differentiation antigens Gene/protein HLA^(a) Peptide PositionSEQ ID NO: CEA A2 YLSGANLNL 605-613 179 A2 IMIGVLVGV 691-699 180 A2GVLVGVALI 694-702 181 A3 HLFGYSWYK  61-69 182 A24 QYSWFVNGTF 268-277 183A24 TYACFVSNL 652-660 184 DR3 AYVCGIQNSVSANRS 568-582 185 DR4DTGFYTLHVIKSDLVNEEATGQFRV 116-140 186 DR4 YSWRINGIPQQHTQV 625-639 187DR7 TYYRPGVNLSLSC 425-437 188 DR7 EIIYPNASLLIQN  99-111 189 DR9YACFVSNLATGRNNS 653-667 190 DR11 LWWVNNQSLPVSP 177-189 191 and 355-367DR13 LWWVNNQSLPVSP 177-189 192 and 355-367 DR14 LWWVNNQSLPVSP 177-189193 and 355-367 DR14 EIIYPNASLLIQN  99-111 194 DR14 NSIVKSITVSASG666-678 195 gp100/Pmel17 A2 KTWGQYWQV 154-162 196 A2 (A)MLGTHTMEV177(8)-186 197 A2 ITDQVPFSV 209-217 198 A2 YLEPGPVTA 280-288 199 A2LLDGTATLRL 457-466 200 A2 VLYRYGSFSV 476-485 201 A2 SLADTNSLAV 570-579202 A2 RLMKQDFSV 619-627 203 A2 RLPRIFCSC 639-647 204 A3 LIYRRRLMK614-622 205 A3 ALLAVGATK  17-25 206 A3 IALNFPGSQK  86-95 207 A3ALNFPGSQK  87-95 208 A11 ALNFPGSQK  87-95 209 A24 VYFFLPDHL intron 4 210A32 RTKQLYPEW  40-42 211 and  47-52 A68 HTMEVTVYHR 182-191 212 B7SSPGCQPPA 529-537 213 B35 VPLDCVLYRY 471-480 214 B35 LPHSSSHWL 630-638215 Cw8 SNDGPTLI  71-78 216 DQ6 GRAMLGTHTMEVTVY 175-189 217 DR4WNRQLYPEWTEAQRLD  44-59 218 DR7 TTEWVETTARELPIPEPE 420-437 219 DR7TGRAMLGTHTMEVTVYH 174-190 220 DR53 GRAMLGTHTMEVTVY 175-189 221Kallikrein 4 DP4 SVSESDTIRSISIAS 125-139 222 DR4 LLANGRMPTVLQCVN 155-169223 DR7 RMPTVLQCVNVSVVS 160-174 224 mammaglobin-A A3 PLLENVISK  23-31225 Melan-A/MART-1 A2 (E)AAGIGILTV 26(27)-35 226 A2 ILTVILGVL  32-40 227B35 EAAGIGILTV  26-35 228 B45 AEEAAGIGIL(T) 24-33(34) 229 Cw7RNGYRALMDKS  51-61 230 DQ6 EEAAGIGILTVI  25-36 231 DR1 AAGIGILTVILGVL 27-40 232 DR3 EEAAGIGILTVI  25-36 233 DR4 RNGYRALMDKSLHVGTQCALTRR 51-73 234 DR11 MPREDAHFIYGYPKKGHGHS   1-20 235 DR52KNCEPVVPNAPPAYEKLSAE  91-110 236 NY-BR-1 A2 SLSKILDTV 904-912 237 OA1A24 LYSACFWWL 126-134 238 PSA A2 FLTPKKLQCV 165-174 239 A2 VISNDVCAQV178-187 240 RAB38/NY-MEL-1 A2 VLHWDPETV  50-58 241 TRP-1/gp75 A31MSLQRQFLR alt. ORF 242 DR4 ISPNSVFSQWRVVCDSLEDYD 277-297 243 DR15SLPYWNFATG 245-254 244 TRP-2 A2 SVYDFFVWL 180-188 245 A2 TLDSQVMSL360-368 246 A31 LLGPGRPYR 197-205 247 A33 LLGPGRPYR 197-205 248 Cw8ANDPIFVVL 387-395 249 DR3 QCTEVRADTRPWSGP  60-74 250 DR15 ALPYWNFATG241-250 251 tyrosinase A1 KCDICTDEY 243-251 252 A1 SSDYVIPIGTY 146-156253 A2 MLLAVLYCL   1-9 254 A2 CLLWSFQTSA   8-17 255 A2 YMDGTMSQV 369-377256 A24 AFLPWHRLF 206-214 257 A26 QCSGNFMGF  90-98 258 B35 TPRLPSSADVEF309-320 259 B35 LPSSADVEF 312-320 260 B38 LHHAFVDSIF 388-397 261 B44SEIWRDIDF 192-200 262 DR4 QNILLSNAPLGPQFP  56-70 263 DR4 SYLQDSDPDSFQD450-462 264 DR15 FLLHHAFVDSIFEQWLQRHRP 386-406 265

Shared antigens of the third group are expressed in a wide variety ofnormal tissues and overexpressed in tumors. Because a minimal amount ofpeptide is required for CTL recognition, a low level of expression innormal tissues may mean that autoimmune damage is not incurred. However,this threshold is difficult to define, as is the normal level ofexpression of those genes for each cell type. A list of these is inTable 4.

TABLE 4 Overexpressed antigens Gene HLA^(a) Peptide Position SEQ ID NO:adipophilin A2 SVASTITGV  129-137 266 AIM-2 A1 RSDSGQQARY intron 267ALDH1A1 A2 LLYKLADLI   88-96 268 BCLX (L) A2 YLNDHLEPWI  173-182 269BING-4 A2 CQWGRLWQL ORF2 270 CALCA A2 VLLQAGSLHA   16-25 271 CPSF A2KVHPVIWSL  250-258 272 A2 LMLQNALTTM 1360-1369 273 cyclin D1 A2LLGATCMFV  101-109 274 DR4 NPPSMVAAGSVVAAV  198-212 275 DKK1 A2ALGGHPLLGV   20-29 276 ENAH (hMena) A2 TMNGSKSPV  502-510 277 Ep-CAM A24RYQLDPKFI  173-181 278 EphA3 DR11 DVTFNIICKKCG  356-367 279 EZH2 A2FMVEDETVL  120-128 280 A2 FINDEIFVEL  165-174 281 A24 KYDCFLHPF  291-299282 A24 KYVGIEREM  735-743 283 FGF5 A3 NTYASPRFK  172-176 284 and 204-207 G250/MN/CAIX A2 HLSTAFARV  254-262 285 HER-2/neu A2 KIFGSLAFL 369-377 286 A2 IISAVVGIL  654-662 287 A2 ALCRWGLLL    5-13 288 A2ILHNGAYSL  435-443 289 A2 RLLQETELV  689-697 290 A2 VVLGVVFGI  665-673291 A2 YMIMVKCWMI  952-961 292 A2 HLYQGCQVV   48-56 293 A2 YLVPQQGFFC1023-1032 294 A2 PLQPEQLQV  391-399 295 A2 TLEEITGYL  402-410 296 A2ALIHHNTHL  466-474 297 A2 PLTSIISAV  650-658 298 A3 VLRENTSPK  754-762299 A24 TYLPTNASL   63-71 300 IL13Ralpha2 A2 WLPFGFILI  345-353 301Intestinal carboxyl esterase B7 SPRWWPTCL alt. ORF 302 alpha-fetoproteinA2 GVALQTMKQ  542-550 303 A2 FMNKFIYEI  158-166 304 DR13 QLAVSVILRV 364-373 305 M-CSF B35 LPAVVGLSPGEQEY alt. ORF 306 MCSP DR11VGQDVSVLFRVTGALQ  693-708 307 mdm-2 A2 VLFYLGQY   53-60 308 Meloe A2TLNDECWPA   36-44 309 MMP-2 A2 GLPPDVQRV  560-568 310 MMP-7 A3SLFPNSPKWTSK   96-107 311 MUC1 A2 STAPPVHNV  950-958 312 A2 LLLLTVLTV  12-20 313 DR3 PGSTAPPAHGVT repeated region 314 p53 A2 LLGRNSFEV 264-272 315 A2 RMPEAAPPV   65-73 316 B46 SQKTYQGSY   99-107 317 DP5PGTRVRAMAIYKQ  153-165 318 DR14 HLIRVEGNLRVE  193-204 319 PAX5 A2TLPGYPPHV  311-319 320 PBF B55 CTACRWKKACQR  499-510 321 PRAME A2VLDGLDVLL  100-108 322 A2 SLYSFPEPEA  142-151 323 A2 ALYVDSLFFL  300-309324 A2 SLLQHLIGL  425-433 325 A24 LYVDSLFFL  301-309 326 PSMA A24NYARTEDFF  178-186 327 RAGE-1 A2 LKLSGVVRL  352-360 328 A2 PLPPARNGGL  32-40 329 B7 SPSSNRIRNT   11-20 330 RGS5 A2 LAALPHSCL    5-13 331 A3GLASFKSFLK   74-83 332 RhoC A3 RAGLQVRKNK  176-185 333 RNF43 A2ALWPWLLMA(T) 11-19(20) 334 A24 NSQPVWLCL  721-729 335 RU2AS B7 LPRWPPPQLantisense 336 secernin 1 A2 KMDAEHPEL  196-204 337 SOX10 A2 AWISKPPGV 332-340 338 A2 SAWISKPPGV  331-340 339 STEAP1 A2 MIAVFLPIV  292-300 340A2 HQQYFYKIPILVINK  102-116 341 survivin A2 ELTLGEFLKL   95-104 342Telomerase A2 ILAKFLHWL  540-548 343 A2 RLVDDFLLV  865-873 344 DR7RPGLLGASVLGLDDI  672-686 345 DR11 LTDLQPYMRQFVAHL  766-780 346 VEGF B27SRFGGAVVR —^(i) 347 WT1 A1 TSEKRPFMCAY  317-327 348 A24 CMTWNQMNL 235-243 349 DP5 LSHLQMHSRKH  337-347 350 DR4 KRYFKLSHLQMHSRKH  332-347351

Mutagenesis

After selecting the particular tumor specific epitope, random amino acidsubstitutions are introduced. Oligonucleotide sequences encoding thepeptide epitope are designed and cloned in an appropriate vector.Mutagenesis can be done according to the skill of the ordinary worker ateach amino acid position of the peptide. The mutant may havesubstitutions at 1, 2, 3, 4, 5, 6 or more positions, depending on theparticular epitope.

The positional libraries are designed such that the codon of interest istotally randomized (NNN), resulting in a pool of oligonucleotides whichcontains every given codon sequence. This mutagenesis approach might belikened to a slot machine which contains three positions (a codon) andeach position has the same 4 possibilities (A, C, G, or T). When pulled,there is a 1 in 64 chance of getting any combination of 3. If pulled 100times there is a high probability that every sequence will berepresented (80% certainty, according to a Poisson distribution). Here,the 100 pulls represent 100 bacterial colonies, each containing adifferent mutant agonist peptide-encoding oligonucleotide. When clonedand expressed, each amino acid should be represented in a library of100, with 80% certainty, according to a Poisson distribution. Apositional library can be generated for each position (amino acid) ofthe target peptide. The APL minigene constructs are fused to a6×-histidine tag (SEQ ID NO: 415), and can easily be separated frombacterial proteins on Co²⁺-coated paramagnetic beads.

The mutagenized epitopes are preferentially expressed in cells as partof an expression vector, more preferentially as a fusion protein. Thepreferred host for the expression vector is bacterial, e.g., a strain ofE. coli. Most preferred is an inducible expression system.

A mutant library is generated using the expression vector in the hostcell. Preferentially, the library is distributed in liquid culture, mostpreferentially in 96 well plates. The cells accumulate a recombinantprotein comprising the sequence of the mutagenized epitopes.

The recombinant protein is released and separated from the host cells.This can be done by lysing the cells to release the recombinant protein.Preferentially, the mutagenized epitope is separated from other cellularproteins by adding protein binding magnetic beads (e.g. 6×-histidine(SEQ ID NO: 415) specific magnetic beads) to cell lysates.

Screening

Initial screens can be done by combining beads containing recombinantmutagenized epitopes with dendritic cells and epitope-specific T cellsand assaying for the production appropriate cytokines, including, butnot limited to, interferon γ, interleukin-4, interleukin-10, andgranulocyte macrophage colony-stimulating factor. That is, APLs arescreened for the ability to activate epitope-specific T cell clonesfollowing cross-presentation of the bead-bound ligand on class I orclass II MHC molecules by dendritic cells (DC).

Attempts by others to measure the functional avidity of tumorepitope-specific CTL generated via unmodified peptide with CTL generatedvia the analogs, have been hampered by the inability to generateCD8⁺/MART-1₂₆₋₃₅-tetramer positive T cell populations using a peptidehaving the natural amino acid sequence of the epitope. Using the methodsdescribed herein, the superagonist APLs elicit differentantigen-specific CTL responses from patient to patient, and that the CTLpopulations generated by APL stimulation are capable of effectivelykilling tumors. Thus these agonist APLs might be considered“conditional” superagonist ligands. Using unique tumor epitope-specificCTL clones in the initial screen that other potential superagonistpeptides can be identified. Panels of potential tumor-associatedsuperagonist peptides may be assembled, to ensure that one or more APLsare effective at generating potent anti-tumor CTL responses from a givenpatient.

Efficacy

To determine how well the identified agonist APLs could prospectivelygenerate tumor epitope-specific CTL populations from peripheral bloodmononuclear cell (PBMC) of tumor patients, the APLs were used tostimulate different patient PBMC samples under standard in vitroconditions. Preferentially, cultures of PBMC are treated with themutagenized epitope and incubated for at least one week. CTLs canreadily be measured using ordinary methods. For example, cells can bestained with FITC-labeled anti-CD8 antibodies and APC-labeledHLA-matched complexes and analyzed by flow cytometry.

The ability of an APL to generate CD4 T cells from PBMC of tumorpatients is also a measure of the efficacy of the mutagenized epitope.

It may be necessary to probe a panel of APLs since the ability of asingle APL to stimulate cells of every patient having the specific tumorcannot be assumed at the outset of measurements.

One aspect of the utility of the APLs lies in their ability to stimulateT cells of a cancer patient ex vivo or in vivo. The stimulated T cellsare effector and regulator CD4⁺ cells, including Th1, Th2, Th9 and/orTh17 cells. The stimulation can involve use of the APLs as purifiedpeptides, or as intracellular products of APL minigenes. APL minigenesmay also be expressed as a string of beads, i.e., multiple CTL geneswithin the same expression vector, or as part of a T helper protein asdescribed in Fomsgaard et al., 1999 Vaccine 18:681-91; Ann et al., 1997J Virol 1192-302; Toes et al., 1997 Proc Natl Acad Sci 94:14660-65; Gaoet al., 2006 Vaccine 24:5491-97, hereby incorporated by reference intheir entirety.

The potential use for these novel antigenic peptides includes their usein anti-tumor vaccine studies; use in adoptive immunotherapy to generatea wider array of anti-tumor CD4⁺ T cell clonotypes; the ability to alterthe phenotype of T regulatory cells in order to more effectivelyactivate anti-tumor CD4⁺ T cells.

EXAMPLES Example 1

Oligonucleotides were designed to have a complimentary 5′ KpnI site anda complimentary 3′ PstI site. The sequences of the saturationmutagenesis sense strands of the MART-1₂₆₋₃₅ positional oligonucleotidesare shown in Table 5 (each sense strand has a corresponding mutantantisense strand):

TABLE 5 MART- SEQ 1₂₆₋₃₅ ID Sense strand of MART-1₂₆₋₃₅ positionalLibrary NO: Saturation Mutagenesis Oligonucleotides P1 352CATCGAGGGAAGGNNNCTCGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA P2 353CATCGAGGGAAGGGAGNNNGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA P3 354CATCGAGGGAAGGCAGCTCNNNGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA P4 355CATCGAGGGAAGGCAGCTCGCCNNNATCGGCATTCTGACCGTTTAATGAATTCTGCA P5 356CATCGAGGGAAGGCAGCTCGCCGGANNNGGCATTCTGACCGTTTAATGAATTCTGCA P6 357CATCGAGGGAAGGCAGCTCGCCGGAATCNNNATTCTGACCGTTTAATGAATTCTGCA P7 358CATCGAGGGAAGGCAGCTCGCCGGAATCGGCNNNCTGACCGTTTAATGAATTCTGCA P8 359CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTNNNACCGTTTAATGAATTCTGCA P9 360CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTCTGNNNGTTTAATGAATTCTGCANNN represents totally randomized codons, any one of sixty-four codons.In a given positional library consisting of 100 mutant oligonucleotidepairings, each codon has high likelihood of being represented.

Variant polypeptide sequences are listed in Table 6.

TABLE 6 Designation Sequence SEQ ID NO Mart-1 EAAGIGILTV 228 A27LELAGIGILTV 362 E26G GLAGIGILTV 363 E26S SLAGIGILTV 364 L33M ELAGIGIMTV365

Similarly, nucleotides encoding variant sequences of NY-ESO-1₁₅₇₋₁₇₀(SEQ ID NO:144) were synthesized that encoded the following sequences(Table 7).

TABLE 7 Designation Sequence SEQ ID NO: NY-ESO-1₁₅₇₋₁₆₅ WT SLLMWITQC 366NY-ESO-1₁₅₇₋₁₇₀ WT SLLMWITQCFLPVF 144 W161I (NYII-51) SLLMIITQCFLPVF 368W161F SLLMFITQCFLPVF 369 I162R SLLMWRTQCFLPVF 370 I162M SLLMWMTQCFLPVF371 I162Q (NYII-6Q) SLLMWQTQCFLPVF 372 1162V SLLMWVTQCFLPVF 373Q164S (NYII-8S) SLLMWITSCFLPVF 374 F170W (NYII-14W) SLLMWITQCFLPVW 375NY-ESO-1 C165V SLLMWITQVF 376Synthetic polypeptides having these sequences were suspended in DMSO.

Example 2

The saturation mutagenesis oligonucleotides were cloned into theexpression vector pQE40 (Qiagen). The plasmids were transformed into E.coli (M15 pREP). Mini-gene products were expressed as fusion proteinscontaining 6×-histidine tags (SEQ ID NO: 415). Following recombinantprotein induction, bacteria were lysed with 8M Urea, pH 8.0. Lysate washarvested and applied to Mg²⁺ coated paramagnetic beads (Talon beads,Dynal), which bind specifically to 6×-histidine (SEQ ID NO: 415).

For saturation mutagenesis libraries, bacterial clones were culturedindividually in wells of 96-well plates.

Melanoma cell lines A375 and MeI 526, CTL clones and the TAP-deficientcell line T2 were maintained in RPMI 1640, containing 25 mM HEPES, 2 mML-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin and 10% humanserum from normal donors. Dendritic cells were prepared from adherentmonocytes, isolated from the PBMC of HLA-A2⁺ healthy donors. IL-4 (500U/mL; R&D Systems, Minneapolis, Minn.) and GM-CSF (800 U/mL; Amgen,Thousand Oaks, Calif.) were added to the monocytes to promote theirdifferentiation into dendritic cells. MART-1₂₆₋₃₅₋specific CTL cloneswere generated as described by Li et al., 2005. J Immunol 175:2261-69,hereby incorporated by reference in its entirety. PBMC used in thisstudy were obtained from HLA-A2⁺ melanoma patients.

Example 3 Saturation Mutagenesis APL Screen

Following the isolation of the recombinant mini-gene APL products onTalon beads, the bead-bound products were “fed” to 100,000 immaturedendritic cells. Following a 4-hour incubation at 37° C., 100,000MART-1₂₆₋₃₅-specific CTL clones were added to DC/bead preparations.Following a 12-hour incubation at 37° C., the supernatant was harvestedand assayed for the concentration of IFN-γ induced by the APL clones.Anti-IFN-γ antibodies (Endogen) used in the sandwich ELISA were used at1 μg/ml in PBS/0.1% BSA.

Variant MART-1₂₆₋₃₅ agonist peptides identified using mutagenesis APLscreening and their corresponding DNA sequences are shown in Table 8.

TABLE 8 Amino Acid SEQ ID Designation Sequence SEQ ID NO DNA Sequence NOMART-1₂₆₋₃₅ EAAGIGILTV 228 NA A27L ELAGIGILTV 362 NA E26G GLAGIGILTV 363ggactcgccggaatcggcattctgacc 377 E265 SLAGIGILTV 364tcactcgccggaatcggcattctgacc 378 E26S SLAGIGILTV 364tcgctcgccggaatcggcattctgacc 379 E265 SLAGIGILTV 364agtctcgccggaatcggcattctgacc 380 L33M ELAGIGIMTV 365gagctcgccggaatcggcatgctgacc 381

Example 4 In Vitro PBMC Stimulations with Analog Peptides and TetramerStaining

On day 0, monocyte-derived dendritic cells were pulsed with 1 μM of eachMART-1₂₆₋₃₅ analog peptide for 2 hours at 37° C. The DCs were washed andadded to 500,000 HLA-A2⁺ PBMC from melanoma patients at a 1:20 ratio in24-well plates. On day 2, 12.5 U/ml of IL-2, 5 ng/ml IL-7, 1 ng/mlIL-15, and 10 ng/ml of IL-21 were added to each culture. Cytokines werereplenished every 2-3 days for 1-week. Following the 1-week primarystimulation, cultures were re-stimulated with 1×10⁶ irradiated monocytespulsed with 10 μM of the peptide used in the primary stimulation. IL-2,IL-7 and IL-15 were added to secondary stimulations on day 2. Cytokineswere replenished every 2-3 days. 500,000 cells from each culture werestained with APC-labeled anti-CD8 antibody (Caltag Lab, Burlingame,Calif.) and PE-labeled MART-1₇₆₋₃₅ HLA-A2.1 tetramers. Stained cellswere analyzed using FACScalibur flow cytometer and CellQuest (BDPharMingen) and analyzed using FlowJo software v8.5 (Tree Star, SanCarlos, Calif.). Cells were stained with tetramers in 25 μl of 2%FCS/BSA for 1 hour at room temperature, followed by anti-CD8 antibodyfor 15 minutes at 4° C.

Example 5 Generation of MART-1₂₆₋₃₅ Polyclonal Cell Lines

Following in vitro peptide stimulation of HLA-A2⁺ PBMC from melanomapatients MelPt-B, MelPt-C, MelPt-D, MelPt-F and a healthy donor(Healthy-1) MART-1₂₆₋₃₅ tetramer and CD8 positive cells were sorted andisolated on BD FACSaria. Isolated cells were replicated using 30 ng/mlanti-CD3 antibody (OKT3) and IL-2 at 50 U/ml in the presence ofirradiated feeder PBMC and LCL for 2 weeks. IL-2 was replenished every2-3 days. Following the stimulation, cultures were stained for thegeneration of MART-1₂₆₋₃₅ tetramer and CD8 positive cell populations.The polyclonal cell lines were tested for lytic activity and TCR Vβusage (MelPt-C only), as described in Example 6.

Example 6 In Vitro Cytotoxicity Assay

Target cells were labeled with 100 μCi of ⁵¹Cr and co-cultured witheffector cells for 4 hours at 37° C. plus 5% CO₂. Targets were melanomacell lines A375 (HLA-A2⁺/NY-ESO-1⁺) and Mel 526 (HLA-A2⁺/MART-1⁺), andT2 cells pulsed with 1 μM of MART-1₂₆₋₃₅ (positive control) orNY-ESO-1₁₅₇₋₁₆₅ (negative control). Effector cells wereMART-1₂₆₋₃₅-tetramer positive polyclonal cell lines generated witheither A27L, E26S, or L33M peptides (SEQ ID NOS:362, 364, and 365,respectively). Assays were performed in triplicate at a 50:1, 25:1 or12.5:1 effector to target ratio. Released ⁵¹Cr was measured with a gammascintillation counter and percent specific lysis was determined by usingthe formula: percent specific release=(experimental release-spontaneousrelease)/(maximum release-spontaneous release).

Example 7 TCR Spectratype Analysis

TCR Vβ spectratype analysis was carried out by the Immune MonitoringLaboratory at Fred Hutchinson Cancer Research Center. Briefly, cDNA wasgenerated from 1×10⁶ MART-1₂₆₋₃₅ tetramer staining polyclonal celllines. Multiplex Vβ PCR primers were then used to amplify the variableregions of the complementarity-determining region 3 (CDR3) of the TCR βchain. Sequence analysis to determine the Vβ usage of the TCRs wasconducted with GenScan.

Example 8 Mart-1₂₆₋₃₅ Specific CTL Clones can Detect Enhanced CTLEpitopes as Reflected by IFN-γ Expression

To identify superagonist APLs, a novel genetic system was discovered anddeveloped. This system employs saturation mutagenesis of agonistpeptide-encoding oligonucleotides, which when expressed in E. coli willcontain position specific single amino acid substitutions. Thepositional libraries are designed such that the codon of interest istotally randomized (NNN), resulting in a pool of oligonucleotides whichcontains every given codon sequence. This mutagenesis approach might belikened to a slot machine which contains three positions (a codon) andeach position has the same 4 possibilities (A, C, G or T). When pulled,there is a 1 in 64 chance of getting any combination of 3. If pulled 100times there is a high probability that every sequence will berepresented (80% certainty, according to a Poisson distribution). Here,the 100 pulls represent 100 bacterial colonies, each containing adifferent mutant agonist peptide-encoding oligonucleotide. When clonedand expressed, each amino acid should be represented in a library of100, with 80% certainty, according to a Poisson distribution. Apositional library can be generated for each position (amino acid) ofthe target peptide. The APL min-gene constructs are fused to a6×-histidine tag (SEQ ID NO: 415), and can easily be separated frombacterial proteins on Co²⁺-coated paramagnetic beads. APLs are screenedfor the ability to activate epitope-specific CTL clones followingcross-presentation of the bead-bound ligand on class I MHC molecules byimmature dendritic cells (DC).

To validate this system and to verify that it was sensitive enough todetect our model tumor-associated HLA-A2 restricted antigenic peptide,MART-1₂₆₋₃₅, as well as an APL superagonist epitope of MART-1₂₆₋₃₅,called MART-1₂₆₋₃₅A27L (henceforward referred to as A27L (SEQ IDNO:362)), oligonucleotides encoding the appropriate peptide sequenceswere cloned, expressed and assayed for the ability to activate antigenspecific CTL clones as described in materials and methods. The CTL cloneused in this assay, called M26-H1, is specific for MART-1₂₆₋₃₅, andexpresses IFN-γ in response to HLA-A2/MART-1₂₆₋₃₅ complexes. Here, theIFN-γ response elicited by the recombinant unmodified MART-1₂₆₋₃₅cross-presented construct is significantly higher than that elicited bythe HLA-A2 restricted negative control, NYESO-1₁₅₇₋₁₆₅ (FIG. 1).Further, the IFN-γ response elicited by the recombinant superagonistAPL, A27L, was more than 2-fold higher than that elicited by therecombinant wild type construct. Yet, the activation of M26-H1 by theunmodified MART-1₂₆₋₃₅ construct was clearly distinguishable from thatelicited by the HLA-A2 restricted negative control construct,NYESO1₁₅₇₋₁₆₅. These results suggest that the HLA-A2 cross-presentedrecombinant ligands are sufficient to elicit detectable antigen-specificresponses from CTL clones, and also that superagonist APLs can bedistinguished based on an increase in IFN-γ expression, relative to thewild type CTL ligand.

Example 9 Saturation Mutagenesis can Effectively Generate Random AminoAcids in the Parental Antigenic Peptide from which Enhanced Agonist APLScan be Identified

The saturation mutagenesis APL library screen depends on 200 μlbacterial expression cultures in 96-well plates. FIG. 1 shows thatcross-presented recombinant ligands can be detected by antigen-specificCTL. However, in that experiment recombinant proteins were produced athigh concentrations in 5 ml cultures. To determine whether therecombinant protein produced in these significantly smaller cultureswould be sufficient to reflect detectable and varying degrees ofactivation, a position 2 (P2) library of MART-1₂₆₋₃₅ (EXAGIGILTV (SEQ IDNO: 416)) was constructed. By screening this library, in addition todetermining if 200 μl cultures produce sufficient concentrations ofrecombinant protein previously identified superagonist APLs, includingA27L could be identified from among 88 unique mutant APL clones. The P2library screen (FIG. 2), using the CTL clone M26-H1, clearly shows thatthe wild type recombinant ligand MART-1₂₆₋₃₅ elicits significantly moreIFN-γ than the negative control. Furthermore, the APL clones from thelibrary that contained leucine residues at P2 (A27L), elicitedsignificantly more IFN-γ expression in comparison to the wild typeligand. Amino acid content was determined from replicated glycerol stockof the P2 bacterial library. Interestingly, APL clones containingmethionine residues at P2 also elicited greater IFN-γ expression thanwild type MART-1₂₆₋₃₅, although not as great as that elicited by theleucine containing APLs, A27L. Like A27L, A27M is a superagonist APL ofMART-1₂₆₋₃₅. Thus, 200 μl bacterial cultures produce sufficientconcentrations of the recombinant ligands to be detected in this screen.Also, superagonist APLs can be identified in a library of at least 88unique APL clones.

Example 10 Putative Enhanced CTL Epitopes of Mart-1₂₆₋₃₅A27L areIdentified in APL Library Screens

On the basis of previous results demonstrating that superagonist APLscan be identified and defined using the saturation mutagenesis screen,remaining positional libraries of MART-1₂₆₋₃₅, were screened usingsimilar methods. Because a potent superagonist APL of MART-1₂₆₋₃₅ hasalready been identified in A27L, A27L was used as the basis for amutational strategy. That is, leucine in position 2 was constant, whileother positions were mutated independently. This would allowsuperagonist APLs to be identified that are more effective than A27L.

The APL libraries were screened with two different high avidityMART-1₂₆₋₃₅-specific CTL clones. A high avidity TCR is defined as havingthe ability to recognize tumor cells that express both MART-1 and HLA-A2class I molecules. The vast majority of the MART-1₂₆₋₃₅ derivativemutant peptide clones screened from each of the positional librarieswere not as effective as A27L at activating the MART-1₂₆₋₃₅₋specific CTLclone (FIG. 3). However, several clones from the P1, P3 and P8 librariesappeared to work similarly as well as the A27L recombinant construct.The initial screen was conducted by screening two unique APL libraryclones simultaneously in a single well. While this approach allows twiceas many APL clones to be screened, the potency of any agonist APL in thepool is potentially underestimated in the initial screen.

Agonist candidates were selected and re-screened based on their abilityto elicit more or comparable levels of IFN-γ from M26-H1 in the initialscreen (FIG. 3B). When tested independently, both of the clones from theP3 libraries elicited less IFN-γ expression from theMART-1₂₆₋₃₅-specific CTL clone, relative to A27L. When re-screenedindependently, it was apparent that only one of the two mutant peptideclones from the P1 and P8 wells was responsible for the increased IFN-γexpression. The DNA encoding these putative MART-1₂₆₋₃₅ agonist peptideswas prepared from the duplicated bacterial glycerol stocks. Theenhancing mutations for the P1 putative agonists contained eitherglycine (E26G) (SEQ ID NO:363) or serine (E26S) (SEQ ID NO:364) residuesat P1 instead of the naturally occurring glutamate residue. The P8putative agonist contained a methionine residue (L33M) (SEQ ID NO:365)at position 8 rather than the naturally occurring leucine residue. Noadditional putative agonists were identified from the library screensusing the second CTL clone, M26-H2.

Example 11 MART-1₂₆₋₃₅ Agonist Peptides Display a Differential Capacityto Activate Different MART-1₂₆₋₃₅₋specific CTL Clones

To analyze the putative superagonist APLs on a molar basis, individualpeptides were synthesized at greater than 90% purity. To determinewhether these APLs would be similarly recognized by uniqueMART-1₂₆₋₃₅-specific CTL clones, the APLs were tested against fourclones bearing unique T cell receptors (TCR). These included two highavidity CTL clones (M26-H1 and M26-H2) and two low avidity CTL clones(M26-L1 and M26-L2) (FIG. 4). Low-avidity TCR is here defined as havingthe ability to respond HLA-A2 positive peptide-pulsed target cells butnot to cells displaying naturally processed and presented determinantsfrom HLA-A2/MART-1 positive tumors. Low-avidity T cells have thepotential to mediate antigen-specific cell and tissue destruction.

FIG. 4 panel A shows that each of the newly identified agonist peptidesis similarly effective in activating M26-H1—the high-avidity CTL cloneused in the initial screen (as described in FIG. 3) as compared toMART-1₂₆₋₃₅ superagonist peptide, A27L. A similar pattern of activationwas found when the identified agonist peptides are used to stimulate theCTL clone M26-H2. In contrast to the above results, the low-avidityMART-1₂₆₋₃₅-specific CTL clones yielded widely divergent results inresponse to different agonist peptides. For example, while the CTL cloneM26-L1 recognizes the peptide E26S more than 100-fold better than A27L(based on half-maximal activation), the CTL clone M26-L2 recognizes A27Lbetter than it does E26S. Similarly, while L33M is scarcely recognizedby the CTL clone M26-L1, it is the most effective agonist for activatingM26-L2. Thus, these analogs might be considered “conditional” agonists,as they do not elicit generalized patterns of activation among uniqueantigen-specific clonotypes.

Example 12 MART-1₂₆₋₃₅ APLs Demonstrate Patient-Specific EnhancedGeneration of MART-1₂₆₋₃₅ CTL Populations from the PBMC of MelanomaPatient Donors

To determine how well the identified agonist APLs could prospectivelygenerate MART-1₂₆₋₃₅-specific CTL populations from melanoma patientperipheral blood mononuclear cell (PBMC) preparations, the APLs wereused to stimulate eight different patient PBMC samples under standard invitro conditions (Table 9).

TABLE 9 Patient A27L E26G E26S L33M MelPt-A 3.14 (1) 1.68 (0.53) 3.36(1.07) 0.98 (0.31) MelPt-B 2.97 (1) 1.31 (0.44)  4.3 (1.45)  7.7 (2.6)MelPt-C 40.6 (1) 45.6 (1.12) 15.6 (0.38) 41.1 (1.02) MelPt-D 0.65 (1)1.73 (2.66) 3.43 (5.27) 2.07 (3.1) MelPt-E 1.77 (1) 8.42 (4.75) 6.88(3.88) 24.2 (13.67) MelPt-F 5.45 (1) 3.35 (0.61) 3.72 (0.68) 3.07 (0.56)MelPt-G 33.4 (1) 1.89 (.06) 1.75 (.05) 2.37 (.07) MelPt-H 1.24 (1) 2.03(1.63) 1.31 (1.06) 2.77 (2.2)

These results show that MART-1₂₆₋₃₅ APLs exhibit differential capacitiesto generate MART-1₂₆₋₃₅-specific CTL populations from the PBMC ofdifferent melanoma patient donors. APLs were used to stimulate PBMCcultures in vitro. Following a one-week secondary stimulation cells werestained with FITC-labeled anti-CD8 antibodies and APC-labeledHLA-A2/MART-1₂₆₋₃₅ tetramers and analyzed by flow cytometry. Values aregiven as percent tetramer positive relative to a negative control. Thefold difference relative to A27L is indicated in parentheses.Differences of more than two-fold are indicated in bold.

One week following the second in vitro stimulation, cultures werestained with the wild-type MART-1₂₆₋₃₅/HLA-A2 tetramer. Similar to theobservations made using different MART-1₂₆₋₃₅-specific CTL clones, noneof the peptide ligands were universally effective in generatingMART-1₂₆₋₃₅-specific CTL populations from all patient PBMC samples (FIG.5). Any given APL was more or less effective in generatingantigen-specific CTL from any given patient PBMC sample. For example,while the agonist peptide E26S is the least effective at generatingMART-1₂₆₋₃₅-specific CD8 positive populations from the PBMC of MelPt-C(3-fold<A27L), it is the most effective APL for generating such T cellpopulations from MelPt-D (5-fold>A27L) Similarly, whereas the agonistpeptide L33M is 14-fold more effective than A27L in generating ofMART-1₂₆₋₃₅-specific CD8 positive populations from the PBMC of MelPt-E,it is 14-fold less effective than A27L in generatingMART-1₂₆₋₃₅-specific CD8 populations from the PBMC of MelPt-G. Thesefindings demonstrate that any one CTL ligand may not be effective atgenerating antigen-specific CTL populations from the PBMC of any givenpatient; and show the importance of establishing a panel of potentialsuperagonist APLs.

Example 13 CD8 Positive MART-1₂₆₋₃₅-Specific Polyclonal Cell LinesGenerated with the Identified MART-1₂₆₋₃₅ Agonist APLS can Kill HLA-A2⁺Tumors Expressing Endogenous MART-1

The use of altered peptide ligands poses the risk of generatingantigen-specific T cells which display relatively low anti-tumorfunctional avidity. To determine whether the MART-1₂₆₋₃₅-specific CTLthat were generated with these novel MART-1₂₆₋₃₅ agonist peptides wereof sufficient functional avidity to kill HLA-A2/MART-1 positive tumortargets, polyclonal lines of CD8 positive MART-1₂₆₋₃₅ tetramer-stainingcells were established from the PBMC of MelPt-B, MelPt-C, MelPt-D,MelPt-F or a healthy donor (Healthy1), stimulated with either A27L, E26Sor L33M agonist peptides (SEQ ID NOS:362, 364, and 365, respectively).These cell lines were screened for reactivity to unmodified MART-1₂₆₋₃₅peptide pulsed HLA-A2 positive targets and to HLA-A2/MART-1 positivetumor targets at varying effector to target ratios in a standardchromium release assay (Table 10).

TABLE 10 Tumor specific lysis by CTL generated with MART₁₂₆₋₃₅ peptideanalogs Percentage Specific Lysis from polyclonal CTL lines generatedwith the indicated peptide MART-1₂₆₋₃₅A27L MART-1₂₆₋₃₅E26SMART-1₂₆₋₃₅L33M T2 + T2 + T2 + Patient E/T T2 M26 A375 Mel526 T2 M26A375 Mel526 T2 M26 A375 Mel526 MelPt-B 50 ND ND ND ND 0^(c) 40 6 58 2 6313 80 25 ND ND ND ND 0 31 4 45 2 52 12 70 12.5 ND ND ND ND 2 21 8 30 441 9 58 MelPt-C 50 9 73 3 35 2 87 8 41 9 91 0 58 25 10 52 5 30 7 66 5 3212 84 2 52 12.5 9 42 2 25 1 54 2 25 11 76 5 45 MelPt-D 50 0 50 10 32 387 8 41 14 90 2 57 25 2 42 10 30 8 65 7 32 11 84 4 54 12.5 5 35 9 25 154 6 25 8 75 6 45 MelPt-F 50 6 92 5 65 ND ND ND ND 23 70 2 44 25 3 81 550 ND ND ND ND 21 72 3 42 12.5 2 73 5 43 ND ND ND ND 22 65 5 38 Healthy150 2 52 3 58 0 34 9 28 2 57 13 65 25 4 42 7 42 0 26 5 15 6 49 13 57 12.51 31 6 35 0 18 8 10 5 38 10 44“ND” is not done. T2 is a TAP-deficient cell line that expressespeptide-unbound HLA-A2 molecules unless pulsed extracellularly. Here, T2was pulsed with NY-ESO-1₁₅₇₋₁₆₅ unless indicated otherwise. M26 is anabbreviation for the unmodified MART-1₂₆₋₃₅ peptide. Numbers representthe percentage specific lysis obtained from each target. T375 is aHLA-A2 positive/MART-1 negative cell line.

The results illustrate that the CTL populations that were generated fromeach PBMC source with either of the altered peptide ligands can killtargets that display wild-type MART-1₂₅₋₃₅ in the context of HLA-A2, andrecognize the epitope with sufficient affinity to kill tumors expressingMART-1.

To determine whether unique or shared MART-1₂₅₋₃₅₋specific CTLclonotypes were generated with each of the peptide ligands (A27L, E26Sand L33M), spectratype analysis was performed on CTL lines derived fromMelPt-C PBMC to determine their Vβ TCR usage. Results showed that theagonist peptides A27L, E26S and L33M generated CTL populations thatprimarily (>90%) utilized TCR Vβ24, Vβ8 and Vβ3, respectively. Thissuggests that the different analog peptides preferentially generatespecific TCR utilizing CTL subsets. Taken together, these resultsdemonstrate the ability of the identified APLs to elicitMART-1₂₆₋₃₅-specific CTL responses that are capable of directly killingMART-1 expressing tumors, and suggest that unique MART-1₂₅₋₃₅-specificTCR subpopulations are being preferentially generated by the differentMART-1₂₆₋₃₅ analog peptides.

Example 14 NY-ESO APLs

The methods of Examples 2-8 were used to generate enhanced agonist APLs.Results of a library screen are shown in FIG. 6. Clones showing activitywere sequenced. Variant sequences with the most activity correspond toamino acid sequences of SEQ ID NOS:368-376.

Using the methods of Example 11 to analyze the putative superagonistAPLs on a molar basis, individual peptides were synthesized at greaterthan 90% purity. To determine whether these APLs would be similarlyrecognized by unique NY-ESO-1-specific CTL clones, the APLs were testedagainst ten clones bearing unique TCR. Each of the newly identifiedagonist peptides is similarly effective in activating CTL clones used inthe initial screen in comparison to wild-type NY-ESO-1₁₅₇₋₁₇₀superagonist peptide. Different patterns of stimulation are obtainedwith different CTL clones. Specific CTL clones yielded widely divergentresults in response to different agonist peptides. Similar to resultsobtained with MART superagonist peptides, these NY-ESO-1 analogs mightbe considered “conditional” agonists, as they do not elicit generalizedpatterns of activation among unique antigen-specific clonotypes.

The NY-ESO-1₁₅₇₋₁₆₅ C165V APL SEQ ID NO:376 was compared to wild-typeNY-ESO-I₁₅₇₋₁₆₅ SEQ ID NO:366 in effectively producing CTL from PBMC.FIG. 7 shows that the variant peptide had a higher avidity than the wildtype sequence to a CD-8⁺ population.

FIG. 8 show the ability of several NY-ESO-1APL to stimulate CD-4⁺fractions (FIG. 8A) and PBMC (FIG. 8B) and. Results showed that NY-ESO1APLs I162Q, Q164S, and F170W (SEQ ID NOS:372, 374, and 375,respectively) were the most effective in stimulating CD-4⁺ cells.

NY-ESO-1₁₅₇₋₁₇₀ agonist peptides identified using mutagenesis APL screenand their corresponding DNA sequences are shown in the following Table11.

TABLE 11 SEQ SEQ Amino Acid ID ID Designation Sequence NO DNA SequenceNO NY-ESO-1₁₅₇₋₁₇₀ SLLMWITQCFLPVF 144 NA W161I SLLMIITQCFLPVF 368agcctgctgatgatcattacccagtgctttctgccggtgttttaa 382 W161I SLLMIITQCFLPVF368 agcctgctgatgattattacccagtgctttctgccggtgttttaa 383 Q164SSLLMWITSCFLPVF 374 agcctgctgatgtggattacctcatgctttctgccggtgttttaa 384F170W SLLMWITQCFLPVW 375 agcctgctgatgtggattacccagtgctttctgccggtgttttgg385 W161F SLLMFITQCFLPVF 369agcctgctgatgtttattacccagtgctttctgccggtgttttaa 386 I162R SLLMWRTQCFLPVF370 agcctgctgatgtggaggacccagtgctttctgccggtgttttaa 387 I162MSLLMWMTQCFLPVF 371 agcctgctgatgtggatgacccagtgctttctgccggtgttttaa 388I162Q SLLMWQTQCFLPVF 372 agcctgctgatgtggcaaacccagtgctttctgccggtgttttaa389 I162V SLLMWVTQCFLPVF 373agcctgctgatgtgggtgacccagtgctttctgccggtgttttaa 390 Q164S SLLMWITSCFLPVF374 agcctgctgatgtggattacctcttgctttctgccggtgttttaa 391

What is claimed:
 1. A compound for the treatment of a patient with atumor, wherein the compound comprises a peptide comprising a tumorepitope, wherein the tumor epitope comprises an amino acid substitutionin a tumor antigen, wherein the tumor antigen is SEQ ID NO: 144 or 228,and wherein the compound comprises a sequence selected from the groupconsisting of SEQ ID NOS: 362-365 and 368-376.
 2. The compound of claim1, wherein the peptide is produced by an expression vector in a hostcell in vitro wherein the expression vector comprises a nucleotidesequence selected from the group consisting of SEQ ID NO: 377-391. 3.The compound of claim 2, where the nucleotide sequence is expressed inhuman cells.
 4. The compound of claim 3, wherein the nucleotide sequenceis expressed in cells obtained from a melanoma patient.
 5. The compoundof claim 1, wherein the tumor antigen is SEQ ID NO: 144, and wherein thecompound comprises a sequence selected from the group consisting of SEQID NOS: 368-376.
 6. The compound of claim 5, wherein the peptide isproduced by an expression vector in a host cell in vitro wherein theexpression vector comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 382-391.
 7. The compound of claim 5,wherein the compound comprises a sequence consisting of SEQ ID NOS: 372,374, or
 375. 8. The compound of claim 1, wherein the tumor antigen isSEQ ID NO: 228, and the compound comprises a sequence selected from thegroup consisting of SEQ ID NOS: 362-365.
 9. The compound of claim 8,wherein the peptide is produced by an expression vector in a host cellin vitro wherein the expression vector comprises a nucleotide sequenceselected from the group consisting of SEQ ID NOS: 377-381.
 10. Thecompound of claim 8, wherein the tumor antigen is SEQ ID NO: 228, andthe compound comprises a sequence consisting of SEQ ID NOS: 362, 363 or365.
 11. The compound of claim 1, wherein the wherein the compoundcomprises a multiplicity of sequences selected from the group consistingof SEQ ID NOS: 362-365 and 368-376.
 12. The compound of claim 11,wherein the wherein the compound comprises a multiplicity of sequencesselected from the group consisting of SEQ ID NOS: 362-365.
 13. Thecompound of claim 12, wherein the compound comprises a sequenceconsisting of SEQ ID NOS: 362, 363 or
 365. 14. The compound of claim 11,wherein the wherein the compound comprises a multiplicity of sequencesselected from the group consisting of SEQ ID NOS: 368-376.
 15. Thecompound of claim 14, wherein the compound comprises a sequenceconsisting of SEQ ID NOS: 372, 374, or 375.